Sensitization of bacterial spores to the lethal effects of certain treatments

ABSTRACT

A method for sterilizing, disinfecting or preserving substances, whether fluid or solid, by subjecting them to hydrostatic pressure for a duration sufficient to germinate bacterial spores present in the substances either before or during the usual treatments such as radiation, heating or chemical treatment.

United States Patent John Gannon Clouston SENSITIZATION 0F BACTERIALSPORES TO THE LETIIAL EFFECTS OF CERTAIN TREATMENTS 12 Claims, NoDrawings u.s. Cl 21/2, 21/60, 99/211, 99/219,195/96 Int. Cl A611 l/00Field of Search. 21/1, 2, DIG. 3, 58, 54, 60,101,102;99/211, 219; 195/96References Cited UNITED STATES PATENTS 4/1922 Crowther 21/60 1,711,0974/1929 Kratzer 99/219 1,746,731 2/1930 Koehler... 99/219 2,069,8202/1937 Dodge 21/60 2,356,505 8/1944 Christensen 21/2 2,374,805 5/1945Camelford 21/60 2,899,266 8/1959 Gewalt et a1. 21/58 3,276,840 10/1966Sierra 21/2 3,457,031 7/1969 Linder et a1. 21/58 3,494,722 2/1970 Gray21/54 FOREIGN PATENTS 418,127 10/1934 Great Britain 99/219 PrimaryExaminer-Morris 0. Wolk Assistant Examiner-Joseph T. ZatargaAttorney-Waters, Roditi, Schwartz & Nissen ABSTRACT: A method forsterilizing, disinfecting or preserving substances, whether fluid orsolid, by subjecting them to hydrostatic pressure for a durationsufficient to germinate bacterial spores present in the substanceseither before or dur ing the usual treatments such as radiation, heatingor chemical treatment.

SENSITIZATION OFBACTERIAL SPORES TO THE LETHAL EFFECTS OF CERTAINTREATMENTS This application is a continuation-in-part application of mycopending application Ser. No. 75 I055 filed 8th Aug., 1968, nowabandoned.

BACKGROUND OF THE INVENTION This invention relates to methods forsterilizing, disinfecting and/or preserving fluid or solid substances.It is well known. as for'example, in US. Pat. No. l4l,3006 and US. Pat.No. 206,9820, that germs or bacteria may be killed by the application ofpressure followed by the sudden release of the pressure. In thesepatents it is not the pressure but the release of pressure whichdisrupts the micro-organisms but this method is not efficient for thekilling of bacterial spores. ln. accordance with this invention it hasbeen found that the application of pressure to fluid or solid substancescontaminated with bacterial spores causes germination of the spores,thereby reducing their resistance to sterilizing, disinfecting and/orpreserving treatments. In accordance with the invention knownsterilizing, disinfecting, and/or preserving treatments such as ionizingradiation, ultraviolet radiation, chemicalor gaseous treatment orheating may be employed for killing spores which have beengerminated bythe application of pressure.

As a result of this invention, the quantity of heat, ionizing radiationor ultraviolet radiation, chemicals or any other treatment that proveslethal required to sterilize, disinfect and/or preserve said fluid orsolid substances is reduced.

SUMMARY OF THE INVENTION The invention providesan improved method ofsterilizing, disinfecting and/or preserving fluid or solid substances,wherein bacterial spores are destroyed by germinating them by theapplication of hydrostatic compression, in the pressure range 100 p.s.i.to 20,000 p.s.i. for a period of time depending on the germinatingpressure and temperature employed to the substances prior to or duringper se known sterilizing and/or disinfecting treatments. In .the. caseof heating the maximum effect is obtained by applying pressure beforeheating.

As a result of the pressure application a physiological change isinduced in the bacterial spores, which remain viable, but consequentlyare more susceptible to the usual sterilizing and disinfectingtreatments mentioned above. The physiological change can be termedinitiation-of-germination, initiation or germination as defined by W. G.Murrel (I961). (Symp.

Soc. Gen. Microbiol, 1l,l l0) and is identified according to theproperties listed by L. J. Rode and J. W. Foster (Proc. Natl. AcadSci.(Wash.) l 18).

The fraction ofspores which initiate germination depends on the periodof compression as well as the applied pressure and temperature. Thefraction is defined as the ratio-of the number of surviving sporesresistant to the lethal effects of heat, radiation, ultraviolet lightand chemicals to the initial number of resistant spores in unit ofspores per millilitre or spores per gram or total spore count. The sameresidual population or resistant spores will resultby applying ahigh-pressure for a short time or a lower pressure for a longer time.

The invention can be used for the germination of any species ofbacterial spores of the genus Bacillus and Clostridium. The duration ofthe treatment depends on the pressure and temperature employed forgermination. At ambient temperatures the degree of germination caused bya pressure of 20,000 p.s.i. applied to Bacillus Pumilus spores for 15minutes at 25 C. is 99.9 percent whereas at a pressure of 9,600 p.s.i.85 percent germinate in IS minutes. At a temperature of 48 C. and apressure of 9,600 p.s.i. 99.5 percent ofthe spores germinate in 15minutes.

The method applies to the pH range 1 to 10 and is effective at ambienttemperature and at temperatures less than or greater than ambient, andalso applies to both aerobic and anaerobic conditions.

reached above which the percent germination decreases with increasingtemperature. As the pressure is increased the optimum temperature forgermination increases but for the pressure range specified the optimumtemperature is less than C. At a pressure of 9,600 p.s.i. the optimumtemperature for Bacillus pumilus is 48 C. At a pressure of 3,750 p.s.i.the bptimum temperature for B. cereus is 38 C. whereas at 7,500 p.s.i.the optimum temperature is 42 C. The optimum temperature for Bacillussubtilis is 50 C. at 7,500 p.s.i. and 70 C. at 15,000 p.s.i. Also, thehydraulic fluid used in the compression can be a liquid ora moist gas,or,' the material being treated can itself be the hydraulic fluid.

The present invention can be further improved by the presence ofexogenous compounds inthe form of ionic inorganic, or organic solutes,which enhance the physiological change in the bacterial spore.

Fluid and solids each require a slight modification in the applicationof compression through the difference in handling problems thereof. Eachtreatment is considered below butit will be appreciated that thedescription is exemplary only and the invention is not intended to belimited to the exemplary methods described.

The pressure process applied to liquids contaminated with bacterialsporesinvolves placement of theliquid in a pressure vessel which is asimple piston and cylinder device. Pressure is generated for therequired time by applying sufficient force to the piston to obtain therequired pressure for the required time.

Alternatively, the pressure can be generated using a hydraulicintensifying pump. Several types of pumps suitable for pressurizingfluids to pressures up to 20,000 p.s.i. using inlet-air pressures up top.s.i. are available commercially. The fluid which is to betreated canbe the hydraulic fluid or it can be separated from the hydraulic fluidby means of an elastic membrane such as rubber or plastic material.

The treatment of solid substances involves placing the contaminatedobject in a pressure vessel which is connected to a compressedgas'cylinder and is subjected to the required gas pressure for therequired period of time.

The object is placed in a pressure vessel which is connected to a gascompressing device via a heat exchanger to control the temperatureof thegas in the pressure vessel which will rise dueto the heat ofcompression. The required pressure is maintained for the requiredtime.The method is more effective when the solid is pressurized by a moistgas. At high-pressures a trace of water vapor in the gas or vessel willcondense onto the solid and assist the physiological change initiated bycompression. 1

DESCRIPTION OF THE PREFERRED EMBODIMENTS The invention is furtherillustrated by the following nonlimiting examples:

EXAMPLE I A suspension of Bacillus pumilus spores in distilled water toa concentration of between five and ten x 10 organisms per ml. wascompressed to 18,000 p.s.i. for 270 minutes (sample 1). An equivalent(sample 2) was compressed simultaneously while being irradiated bycobalt 60 gamma rays. A further equivalent sample was maintained atatmospheric pressure and 25 C. and irradiated (sample 3). A controlsample was maintained at 25 C. and one atmosphere but not pressurized orirradiated.

Immediately following decompression, the survival was measured by thedrop count method using oxoid nutrient broth containing 1 percent oxoidagar No. 3 gas as the nutritive medium. After 24 to 48 hours incubationat 37 C. colonies were counted using a Brunswick colony counter. Furtherincubation did not increase the number of colonies. The total viablecount wasestimated from at least two different dilutions by the methodof Farmiloe, F. J. Cornford, S. .1. Coppock, J. B. M. and Ingram, M. .1.Sci. Food Agric. 5, 292,304 (1954).

The number of surviving resistant spores and the number of survivingforms sensitive to the treatment was established by heating the sporedilutions used in estimating the total viable count at 80 C. for 15minutes. After cooling, drop counts were made as described above and thenumber of forms which had initiated germination calculated bydifference.

The results expressed as the number of surviving heat stable sporesinitial number of heat stable spores were as follows:

Sample Medium T t Radiation Dose Result C. p.s.i. min. rads 1 Water 2518,000 270 nil.

2 Water 25 18.000 270 247.000

3 Water 25 14.7 270 247.000

Control 4 25 14.7 270 nil Heating at 80 C. for 15 min. and 14.7 p.s.i.reduced the spore count at most by percent. Compression to 18,000 p.s.i.for 270 min., followed by heating to 80 C. for min. reduced the sporecount by 94.8 percent. Irradiation for 270 min. at 14.7 p.s.i. reducedthe spore count by at most 87 percent. Compression during irradiationreduced the spore count by 99.3 percent.

EXAMPLE 2 A suspension of Bacillus pumilus spores in 0.067M potassiummono and dihydrogen phosphate pI-16.8 was compressed to 16,000 p.s.i.for 240 min. (sample 1). Sample (2) was compressed simultaneously whilebeing irradiated by cobalt 60 gamma rays. Sample (3) was irradiated atatmospheric pressure. Sample (4) the control was maintained at C. andmeasurements made as before.

Results were as follows expressed as above.

Sample Medium T p t Radiation Result C. p.s.i. min. Dose rads I 0.067Mpotas- 25 16.000 240 nil 0.000155 2 sium monoand 25 16.000 40 110.0000.000037 3 dihydrogen 25 14.7 240 220.000 0.143

4 phosphate 25 14 7 40 nil Heating at 80 C. for 15 min. did not alterthe spore count. Compression to 16,000 p.s.i. for 240 min. followed byheating to 80 C. for 15 min. reduced the spore count by 99.985 percent.Irradiation for 240 min. reduced the spore count by at most 86 percent.Irradiation during compression reduced the spore count by 99.996percent.

EXAMPLE 3 LII The result shows that presence of exogenous solutesincreases the rate of decrease of the resistant bacterial spore countand by suitable choice of additive a similar but greater effect can beobtained at a pressure of 6,600 p.s.i. for 30 min. as is obtained by apressure of 16,000 p.s.i. for 30 min.

Compounds such as tyrosine, adenosine. glucose, mannose, I-arginine,l-phenylalanine, l-alanine, 1-cysteine,inosine and yeast extract, whenadded in suitable proportions will initiate germination at ambienttemperature and pressures exceeding 100 p.s.i.

EXAMPLE 4 Effect ofTemperature at Constant Pressure Organism T P lMedium Result C. p.s.i. mins. '1 germination B.subtilis 38.7 7.000 30Potassium phosphate buffer 405 7.000 30 60 41.2 7.000 30 43.0 7,000 30B.subtilis 35.0 8.000 30 Distilled \LLIICI EXAMPLES Effect ofTime ofCompression at Different Temperatures Organism T P 7tgcrm Medium ResultC. inution Time for 50;

germination B.subtilis 32,6 9 280 50 Potassium 60 mins.

bulTer 38.7 9.280 50 30 mins.

40.8 9.280 50 25 mins.

42.) 9.280 50 18 mins.

EXAMPLE 6 Effect ofincreasing pressure at constant temperature andduration of compression Organism T P t Medium Result C. p.s.i. mins. 1germination B.cereusT 25 7.000 10 Potassium phosphate buffer EXAMPLE 7Effect of compression on different organisms Result,

percent T, P, t, germi- Organism C. p.s.i. min. Medium nation B.subtilis 25 15,000 30 Phosphate bufler 90 B.cagulan. 25 15,000 80 B.pumilua 25 15,000 00 Cl. sporoaenea... 25 20,000 70 EXAMPLE 8 Effect ofexogenous compounds on initlatiou or germination by hydro staticpressure at constant temperature iie pafiic ularly useful pui poseapplication of the method of the present invention is in the sterilizingof surgical sutures. The sutures in their final sealed containers may besubjected to hydrostatic pressure and temperatures in the range 25 C. to70C., before or during sterilizing treatment. The method involvesplacing the sutures after manufacture in a container or containers whichmay be metal, plastic or glass containing an aqueous solution. Thecontainer may be flexible or rigid, but if rigid must contain means suchas a diaphragm for transmitting the pressure to the aqueous solutionsurrounding the sutures.

The container is placed in a pressure vessel, the pressure vessel sealedand using water as the hydraulic fluid and conventional methods ofgenerating hydrostatic pressure, the container is compressed bypressures in the range 100 to 20,000 p.s.i. for a period of timedepending on the temperature and the nature of exogenous compound (ifany) in the solution. The container may simultaneously be subjected tothe sterilizing effect of ionizing radiation or subsequently treated byradiation, heat, ultraviolet light or gaseous treatment. One suitablecontainer would be a plastic container filled with the aqueous solutionand sealed with a stopper in such a way that air is excluded and thestopper and solution are in intimate contact. The stopper, especially inthe case of a rigid container, should be of rubber or plastic to ensurethat the hydrostatic pressure is transmitted to the solution in thecontainer.

What i claim is:

1. in a method of sterilizing, disinfecting and/or preserving fluid orsolid substances contaminated with bacterial spores of the genusBacillus and Clostridium. the improvement comprising germinating saidspores by hydrostatic compression applied to said substance in thepressure range of 100 psi. to 20,000 psi. and at a temperature less thanC, said pressure and temperature being maintained for a period of timesufficient to germinate said spores, and either simultaneously with orsubsequently to said germinating step exposing said substance to asterilizing, disinfecting and/or preserving treatment to destroy saidspores.

2. A method according to claim 1 wherein the sterilizing. disinfectingand/or preserving is effected by ionizing radiation.

3. A method according to claim 1 wherein the sterilizing, disinfectingand/or preserving is effected by ultraviolet radiation.

4. A method according to claim 1 wherein the sterilizing, disinfectingand/or preserving is effected by chemical treatment.

5. A method according to claim 1 wherein the sterilizing, disinfectingand/or preserving is effected by gaseous treat ment.

6. A method according to claim I wherein the sterilizing, disinfectingand/or preserving is effected by heating.

7. A method as claimed in claim 6 wherein the hydrostatic compression isapplied before heating.

8. A method as claimed in claim 1 comprising adding exogenous compoundsin the form of ionic inorganic or organic solutes to the substancesbefore they are subjected to the hydrostatic compression.

9. A method as claimed in claim 1 wherein the substance is a liquidsubstance and same is placed in a pressure vessel comprising a pistonand cylinder and the compression is applied by applying sufficient forceto the piston to obtain the desired pressure for the desired time.

10. A method as claimed in claim 1 wherein the substance is a liquidsubstance and the compression is generated by using a hydraulicintensifying pump.

11. A method as claimed in claim 1 wherein the substance is a solidsubstance and the compression is applied by a gas pressure on thesubstance placed in a pressure vessel.

12. A method as claimed in claim 11 wherein the solid is pressurized bya moist gas.

* i t i

2. A method according to claim 1 wherein the sterilizing, disinfectingand/or preserving is effected by ionizing radiation.
 3. A methodaccording to claim 1 wherein the sterilizing, disinfecting and/orpreserving is effected by ultraviolet radiation.
 4. A method accordingto claim 1 wherein the sterilizing, disinfecting and/or preserving iseffected by chemical treatment.
 5. A method according to claim 1 whereinthe sterilizing, disinfecting and/or preserving is effected by gaseoustreatment.
 6. A method according to claim 1 wherein the sterilizing,disinfecting and/or preserving is effected by heating.
 7. A method asclaimed in claim 6 wherein the hydrostatic compression is applied beforeheating.
 8. A method as claimed in claim 1 comprising adding exogenouscompounds in the form of ionic inorganic or organic solutes to thesubstances before they are subjected to the hydrostatic compression. 9.A method as claimed in claim 1 wherein the substance is a liquidsubstance and same is placed in a pressure vessel comprising a pistonand cylinder and the compression is applied by applying sufficient forceto the piston to obtain the desired pressure for the desired time.
 10. Amethod as claimed in claim 1 wherein the substance is a liquid substanceand the compression is generated by using a hydraulic intensifying pump.11. A method as claimed in claim 1 wherein the substance is a solidsubstance and the compression is applied by a gas pressure on thesubstance placed in a pressure vessel.
 12. A method as claimed in claim11 wherein the solid is pressurized by a moist gas.